1. Field of the Invention
The invention relates to the genetic manipulation of non-human animals. More particularly, in some embodiments, the invention relates to selecting α(1,3)-galactosyltransferase (GGTA1) null cells and genetic manipulation of non-human animals to be used for xenotransplantation.
2. Summary of the Related Art
Clinical transplantation has become one of the major treatments for end stage organ failure since the introduction of chronic immunosuppressive drugs in the mid 1980s. This success has brought about the secondary issue of human organ supply, which greatly limits the ability to provide organs to patients in need of transplants. One of the major approaches to solving this medical need is the utilization of alternative species as a source of organs (xenotransplantation). R. W Evans, in Xenotransplantation, J. L. Platt, Ed. (ASM Press, Washington, D.C., 2001), pp. 29-51, teaches that the pig is the primary alternative species due to ethical considerations, breeding characteristics, infectious disease concerns and its compatible size and physiology.
A major barrier to progress in pig-to-primate organ transplantation is the presence of terminal α(1,3)-galactosyl-(gal)-epitopes on the surface of pig cells. Humans and Old World monkeys have lost the corresponding galactosyltransferase activity in the course of evolution and therefore produce preformed natural antibodies against the epitopes that are responsible for hyperacute rejection of porcine organs. The temporary removal of recipient anti-gal antibodies through affinity adsorption and expression of complement regulators in transgenic pigs has allowed survival of pig organs beyond the hyperacute rejection stage. However, D. Lambrigts, D. H. Sachs, D. K. S Cooper, Transplantation 66, 547 (1998), teaches that returning antibody and residual complement activity are likely to be responsible for the acute and delayed damage which severely limits organ survival even in the presence of high levels of immunosuppressive drugs and other clinical intervention. Attempts have also been made to prevent rejection by reducing expression of gal epitopes through genetic engineering of the donor animal. Unfortunately, C. Costa et al., FASEB J. 13, 1762 (1999), discloses that competitive inhibition of galtransferase in H-transferase transgenic pigs results in only partial reduction in epitope numbers. Other similar approaches have been disclosed in Sandrin et al U.S. Pat. No. 5,821,117, Diamond et al U.S. Pat. No. 6,166,288, and Cooper et al U.S. Pat. No. 6,331,658, all teaching methods for masking the gal epitope through the genetically modified increased expression of carbohydrate epitopes. Similarly, S. Miyagawa et al., J. Biol. Chem. 276, 39310 (2001), teaches that attempts to block expression of gal epitopes in N-acetylglucosaminyltransferase III transgenic pigs also results in only partial reduction of gal epitopes numbers and fails to significantly extend graft survival in primate recipients. Given the large number of gal epitopes present on pig cells, it seems unlikely that any dominant transgenic approach of this nature can provide sufficient protection from anti-gal mediated damage.
A. D. Thall, P. Maly, J. B. Lowe, J. Biol. Chem. 270, 21,437 (1995), and D'Aspice et al, U.S. Pat. No. 5,849,991 teach that viable GGTA1 knockout mice can be produced using ES cell technology. K. L. McCreath et al., Nature 405, 1066 (2000), and Denning et al. PCT Publication WO 01/88096 teach that nuclear transfer technology can be used for locus specific modification of certain large animals, as demonstrated by the production of viable sheep using in vitro targeted somatic cells. K. W. Park et al., Anim. Biotech. In press (2001), discloses successful cloning and production of transgenic pigs by nuclear transfer of genetically modified somatic cells. Gustafsson and Sachs, U.S. Pat. No. 6,153,428 (2000), discloses genetically modified porcine cells in vitro in which the GGTA1 gene has been disrupted by homologous recombination. Dai et al, Nature Biotechnol. 20 (3): 251-255 (2002) teaches production of a pig heterozygous for GGTA1 disrupted gene and Lai et al, Science. 295: 1089 (2002) teaches production of a miniature swine heterozygous for GGTA1 disrupted gene. Gustaffson et al. U.S. Pat. No. 6,413,769 teaches the use of a synthetic antisense oligonucleotide (S-oligonucleotides) to create inactivated heterozygous GGTA1 disrupted miniature porcine cells. Gustaffson et al. further teaches the generation of heterozygous miniature swine by using a drug selection system whereby a vector delivers an antibacterial resistant sequence i.e. neomycin resistant, into the genome to render the GGTA1 gene inactivated. Unfortunately, Bondioli et al., Mol. Reproduc. Dev. 60: 189-195 (2001) reports that the attempt to use nuclear transfer technology to accomplish this in pigs in vivo has been unsuccessful. This is further reported by Ioannu et al, PCT publication WO 97/16064 which discloses that a knockout pig cannot be done.
Gustaffson further discloses in U.S. Pat. No. 6,413,769 the possibility of using antisense technology to produce a miniature swine functionally unable to produce α(1,3)-galactosyltransferase and a method for generation of homozygous miniature swine using a second drug selection system such as herpes simplex virus-thymidine kinase (HSV-tk). Unfortunately, he does not demonstrate that a viable homozygous swine was produced using either method and does not demonstrate that cells were able to be produced and validated for GGTA1 disrupted gene.
There is, therefore, a need for viable GGTA1 null swine defined as swine which do not express any GGTA1 epitopes and which include but are not limited to swine having both alleles of the GGTA1 gene disrupted or rendered non-functional; and swine having one copy of the GGTA1 allele instead of the usual two alleles for the GGTA1 gene and the said copy of the GGTA1 allele is disrupted or rendered non-functional. There is also a need for methods for making such GGTA1 null swine; and methods of using the tissues and organs of such GGTA1 null swine for xenotransplantation.